Although Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) expression is prototypic of transcripts regulated by their half-lives, how this occurs is not yet well understood. In 1986, Shaw and Kamen reported that an AU rich element (ARE) in the 3' untranslated region of GM-CSF mRNA confers a short half-life on otherwise stable transcripts. How T lymphocytes, which make GM-CSF, use the ARE to accomplish this destabilization remains unclear. We are addressing this question by developing a cell-free system to assay GM-CSF mRNA half-life. The establishment of a simplified, in vitro method for measuring GM-CSF mRNA stability will help us answer two questions: which nuclease(s) contribute to the decay of this mRNA; and how factors known to regulate the half-life of GM-CSF mRNA in cells, for example Tissue Plasminogen Activator (TPA), which has been shown to increase the half-life of GM-CSF mRNA, carry out this regulatory activity. As mentioned above, the ARE has been identified as an important instability element in GM-CSF mRNA. However, other elements contributing to the short half-life of GM-CSF mRNA probably exist, as the removal of the ARE fails to completely stabilize the construct from which it has been deleted. We are using a genetic/molecular biological approach to identify new RNA elements, possibly in the protein coding region which contribute to the instability of GM-CSF mRNA. The constructs we plan to use are chimeric genes in which we exchange portions of the long-lived globin gene with GM-CSF in an effort to confer GM-CSF-like instability onto the parent globin construct. These constructs will be transfected into a T cell line and the in vivo half-life will be determined using established techniques.